Expression of phospholipid:diacylglycerine acyltranssferase (pdat) for the production of 
plant storage lipids with polyunsaturated fatty acids

ABSTRACT

The present invention relates to the use of an enzyme mixture containing at least one enzyme with phospholipid:diacylglycerol acyltransferase activity for the production of plant storage lipids containing polyunsaturated fatty acids.

The present invention relates to the use of an enzyme mixture containingat least one enzyme with phospholipid:diacylglycerol acyltransferaseactivity for the production of plant storage lipids containingpolyunsaturated fatty acids.

Triacylglycerol (TAG) constitutes the commonest fat-based energy storefound in nature. Besides acyl-CoA:diacylglycerol acyltransferases(DAGAT), phospholipid:diacylglycerol acryltransferases (PDATs), whichcatalyze the synthesis of storage fats (triacylglycerol, TAG), are knownto date (Dahlqvist et al., Proc. Natl. Acad. Sci., USA, 2000; 97:6487-6492). The enzymes with PDAT activity catalyze, in anacyl-CoA-independent reaction, the transfer of acyl groups from the sn-2position of the phospholipid to diacylglycerol (DAG), thus giving riseto TAG and a lysophospholipid.

Biochemical studies into this transfer reaction have already beencarried out on seeds of Ricinus communis and Crepis palestina, both ofwhich accumulate a high ricinoleic acid and vernolic acid content,respectively, and in sunflowers, whose seed oil contains only saturatedfatty acids (Dahlqvist et al., Proc. Natl. Acad. Sci., USA, 2000; 97:6487-6492).

In plants such as oilseed rape, sunflower, oil palm and the like, theoil (i.e. triacylglycerol) is the most valuable product of the seeds orfruit. Other constituents, such as starch, protein and fibers, arethought of as by-products of lesser value. Increasing the amount of oilon a weight basis, at the expense of other constituents, in oil plantswould thus increase the value of the plant.

By modifying the activity of the genes which regulate the distributionof reduced carbon to oil production it would be feasible for the cellsto accumulate more oil at the expense of other products. Such genesmight be used not only in cells in which oil production is already high,such as, for example, oil plants, but might also induce substantial oilproduction in plants with a moderate or low oil content, such as, forexample, soybeans, oats, maize, potatoes, sugarbeet or swedes, and inmicroorganisms.

Genes encoding a phospholipid:diacylglycerol acyl-transferase havepreviously been cloned from yeast (WO 00/60095). In yeast, PDAT showsdependency on the polar head groups of the donor lipid, the transferredacyl group, and the acyl chain of the acceptor molecule DAG. Theincreased expression of yeast genes encoding enzymes with PDAT activityin the homologous yeast system itself results in an increased oilcontent in the cells in question. In this context, it is mainly themonounsaturated fatty acids with hydroxyl, epoxy and acetylene groupswhich are removed from the membrane and converted into the storage lipidTAG (WO 00/60095).

WO 00/60095 furthermore also describes two nucleotide sequences ofArabidopsis thaliana. Transferring the Arabidopsis gene into yeastconstituted functional proof for the fact that these Arabidopsis genesencode an enzyme with PDAT activity (WO 00/60095).

However, there is substantial interest worldwide not only inmonounsaturated fatty acids, but also in polyunsaturated fatty acids(PUFAs) for large-scale use. These polyunsaturated fatty acids are ofutmost economic interest for example for supplementing foods and feeds.Thus, a high content in lipids with unsaturated fatty acids,specifically polyunsaturated fatty acids, is important in the nutritionof animals and humans since the former additionally have a positiveeffect on the triglycerol level, or cholesterol level, and thus reducethe risk of heart disease. Unsaturated fatty acids are employed in avariety of dietetic foods or medicaments. Polyunsaturated fatty acidsare essential nutrients since the human and animal organism is notcapable of producing them itself. As a rule, polyunsaturated fatty acidsare, however, not found in plants or if so then only in concentrationswhich are of no economic interest.

It is therefore an object of the present invention to providepolyunsaturated fatty acids from renewable plant resources, avoiding thedisadvantages of traditional production methods such as, for example,production by the complicated fermentation of (microbial) single cells,distillation from fish oil or eco-unfriendly methods from non-renewablepetrochemical products.

This object is achieved by the use of an enzyme mixture comprising atleast one enzyme with phospholipid:diacylglycerol acyltransferase (PDAT)activity for the production of plant storage lipids containingpolyunsaturated fatty acids.

It is furthermore possible, by the use according to the invention of anenzyme mixture containing at least one PDAT enzyme, together with atleast one further enzyme for the synthesis of unusual fatty acids, toprovide for example fatty acids with conjugated double bonds orlong-chain polyunsaturated fatty acids.

“Polyunsaturated fatty acids” are understood as meaning those fattyacids with a chain length of at least 14 carbon atoms which have atleast 3 double bonds. For the purposes of the present inventionpolyunsaturated fatty acids belong to the unusual fatty acids which aregenerally not found in plants.

The “unusual fatty acids” include, for example, fatty acids withhydroxyl, epoxy and acetylene groups, polyunsaturated fatty acids,preferably long-chain polyunsaturated fatty acids or fatty acids withconjugated double bonds. Those of interest are, for example,gamma-linolenic acid, arachidonic acid, stearidonic acid,eicosapentaenoic acid or docosahexaenoic acid, conjugated linolic acidor conjugated linolenic. acid (CLA). However, this enumeration is notlimiting.

In the present invention, long-chain polyunsaturated fatty acids arepreferred among the polyunsaturated fatty acids. “Long-chainpolyunsaturated fatty acids are understood as meaning fatty acids with achain length of at least 18 carbon atoms and at least 3 double bonds.Preferred fatty acids are those with at least 3 double bonds and with18-24, especially preferably 18-22, in particular 20, carbon atoms.These include, for example, arachidonic acid, stearidonic acid,eicosapentaenoic acid, gamma-linolenic acid or docosahexaenoic acid.Arachidonic acid is preferred.

Conjugated fatty acids are understood as meaning fatty acids with atleast 16 carbon atoms and at least 3 conjugated double bonds, such as,for example, CLA (conjugated linolenic acid).

A series of enzymes, such as, for example, hydroxylases, epoxygenases,acetylenases, desaturases, elongases, conjugases, trans-desaturases orisomerases, are involved in the synthesis of these unusual fatty acids.The resulting unusual fatty acids are incorporated into the membranelipids by the plants.

Since unusual fatty acids do not normally occur in plant membranes, themaintenance of correct membrane function, and thus correct cellularfunction, must be ensured. Accordingly, the unusual fatty acids shouldonly be present in low concentrations in the membrane lipids. Thisrequires that the unusual fatty acids, once incorporated in the membranelipids, are again removed therefrom efficiently. In the presentinvention, this is achieved by employing an enzyme mixture containing atleast one enzyme with PDAT activity, the PDAT enzyme removing theunusual fatty acid from the membrane lipids and transporting it to thestorage lipids (TAG) of the plant seeds.

The present invention furthermore encompasses the use of an enzymemixture containing at least one enzyme with phospholipid:diacylglycerolacyltransferase activity and at least one further enzyme with theactivity of a hydroxylase, epoxygenase, acetylenase, desaturase,elongase, conjugase, trans-desaturases or isomerases for producing plantstorage lipids containing polyunsaturated fatty acids.

The coexpression of PDAT and further enzymes which are involved in thesynthesis of unusual fatty acids brings about, in plants, a greateraccumulation of unusual fatty acids in the storage lipids than is thecase without PDAT.

In a preferred variant of the present invention, an enzyme mixturecontaining an enzyme with phospholipid:diacylglycerol acyltransferaseactivity and desaturase activity and elongase activity is used. This usecan thus serve for the production of long-chain polyunsaturated fattyacids. Preferably, these are gamma-linolenic acid, arachidonic acid,eicosapentaenoic acid, stearidonic acid or docosahexaenoic acid.

Also encompassed within the invention is the use of an enzyme mixturecontaining an enzyme with phospholipid:diacylglycerol acyltransferaseactivity and desaturase activity or trans-desaturase activity forproducing gamma-linolenic acid or conjugated linoleic acid. The use ofan enzyme mixture containing at least one enzyme withphospholipid:diacylglycerol acyltransferase activity and one enzyme withconjugase, trans-desaturase or isomerase activity, is suitable inaccordance with the invention for the production of conjugated fattyacids such as, for example, conjugated linolenic acid (CLA).

The synthesis, in plants, of polyunsaturated fatty acids, theirincorporation into the membrane lipids and the conversion into storagelipids of the plant seeds are achieved by increasing at least theactivity of one PDAT enzyme. This can be brought about by increased geneexpression, increased catalytic enzyme activity or modified regulatoryenzyme activity. The skilled worker is familiar with measures requiredfor this purpose.

In accordance with the invention, the expression of a gene encoding aPDAT enzyme together with at least one further gene encoding an enzymeof the group of hydroxylases, epoxygenases, acetylenases, desaturases,elongases, conjugases, trans-desaturases or isomerases is required forthe synthesis of unusual fatty acids, such as long-chain polyunsaturatedor conjugated fatty acids.

Increased expression of a gene can be achieved by a procedure with whichthe skilled worker is familiar. These procedures include, for example,increasing the copy number of the gene in question by at least a factorof 2, advantageously by a factor of 5-10. The replicating nucleotidesequence may be chromosomally or extrachromosomally encoded according tothe invention.

Operative linkage with regulatory sequences may furthermore bementioned. They may influence transcription, RNA stability or RNAprocessing, and translation. Examples of regulatory sequences are, interalia, promoters, enhancers, operators, terminators or translationenhancers. They may take the form of natural regulatory sequences ormodified regulatory sequences. The amplification of regulatory sequencesis also feasible.

Increased gene expression, in this context, is to be viewed with regardto an endogenously (naturally) present enzyme activity. Also encompassedfor the purposes of the present invention is heterologous geneexpression, that is to say the expression of one or more genes which donot naturally occur in plants, in which case increased gene expressionis to be regarded as an increase over a value of zero.

A modified, preferably increased, catalytic and/or modified regulatoryactivity of PDAT enzymes or of further enzymes which are involved in thesynthesis of unusual fatty acids can be undertakes by recombinantmodifications of the coding sequence in question, or by what is known asmolelcular modeling. The measures required for this purpose are standardlaboratory practice for the skilled worker.

What has been said before regarding the increased expression/enzymeactivity also applies to the enzyme(s) which is/are present in a plantcell in addition to phospholipid:diacylglycerol acyltransferase in orderto increasingly convert unusual fatty acids into storage lipids.

In an advantageous embodiment of the present invention, a nucleotidesequence encoding a plant-derived enzyme with PDAT activity is used. Theisolated nucleotide sequence encoding an enzyme with PDAT activity ispreferably derived from Arabidopsis thaliana.

In a further variant of the present invention, the enzyme with PDATactivity encompasses an amino acid sequence as shown in SEQ ID No. 2encoded by a nucleotide sequence as shown in SEQ ID No. 1, or allelesthereof.

These sequences are disclosed in WO 00/60095 as the sequence referred toas AB006704.

In accordance with the invention, an isolated nucleic acid, or anisolated nucleic acid fragment, is understood as meaning an RNA or DNApolymer which can be single- or double-stranded and may optionallycontain natural, chemically synthesized, modified or artificialnucleotides. In this context, the term DNA polymer also includes genomicDNA, cDNA or mixtures of these.

In accordance with the invention, alleles are understood as meaningfunctionally equivalent nucleotide sequences i.e. nucleotide sequenceswith essentially the same action. Functionally equivalent sequences arethose sequences which retain the desired function despite a deviatingnucleotide sequence, for example owing to the degeneracy of the geneticcode. Thus, functional equivalents encompass naturally occurringvariants of the sequences described herein, but also artificialnucleotide sequences, for example nucleotide sequences which have. beenobtained by chemical synthesis and which, if appropriate, have beenadapted to suit the codon usage of the host organism. In addition,functionally equivalent sequences encompass those with a modifiednucleotide sequence which imparts to the enzyme for exampledesensitivity or resistance to inhibitors.

A functional equivalent is also to be understood as meaning, inparticular, natural or artificial mutations of a sequence which hasoriginally been isolated, which mutations continue to show the desiredfunction. Mutations encompass substitutions, additions, deletions,exchanges or insertions of one or more nucleotide residues. Alsoincluded are what are known as sense mutations, which, at the proteinlevel, for example lead to the substitution of conserved amino acids,but which do not lead to a principal change in the activity of theprotein and are thus neutral with regard to its function. They alsoinclude modifications of the nucleotide sequence which, at the proteinlevel, concern the N- or C terminus of a protein, but without, however,having a major adverse effect on the function of the protein. Indeed,these modifications may have a stabilizing effect on protein structure.

Other nucleotide sequences which are also encompassed by the presentinvention are, for example, those which are obtained by modification ofthe nucleotide sequence, resulting in corresponding derivatives. Thepurpose of such a modification may be, for example, the furtherdelimitation of the coding sequence contained therein, or else, forexample, the insertion of further cleavage sites for restrictionenzymes. Functional equivalents are also those variants whose functionis weakened or increased in comparison with the starting gene, or genefragment.

Also subject of the present invention are artificial DNA sequences, aslong as they impart the desired characteristics, as described above.Such artificial DNA sequences can be identified for example byback-translation of proteins generated by means of computer-aidedprograms (molecular modeling), or by in vitro selection. Especiallysuitable are coding DNA sequences which have been obtained bybacktranslating a polypeptide sequence in accordance with thehost-organism-specific codon usage. The skilled worker who is familiarwith molecular-genetic methods can readily determine the specific codonusage by means of computer evaluations of other, known genes of theorganism to be transformed.

Homologous sequences are understood as meaning, in accordance with theinvention, those which are complementary to the nucleotide sequencesaccording to the invention and/or which hybridize therewith. Inaccordance with the invention, the term hybridizing sequences includessubstantially similar nucleotide sequences from among the group of DNAor RNA, which enter a specific interaction (binding) with the.abovementioned nucleotide sequences under stringent conditions which areknown per se. A preferred, nonlimiting example for stringenthybridization conditions is hybridization in 6× sodium chloride/sodiumcitrate (SSC) at approximately 45° C. followed by one or more wash stepsin 0.2×SSC, 0.1% SDS at 50-65° C. Also included are short nucleotidesequences of, for example, 10 to 30 nucleotides, preferably 12 to 15nucleotides. Primers or hybridization probes are likewise included.

A homologous nucleotide sequence for the purposes of the presentinvention is a sequence which has at least approximately 40%, preferablyat least approximately 50% or 60%, particularly preferably at leastapproximately 70%, 80% or 90% and most preferably at least approximately95%, 96%, 97%, 98% or 99% or more homology with a nucleotide sequence asshown in SEQ ID No. 1.

Also included in accordance with the invention are the sequence regionswhich proceed (5′, or upstream) the coding regions (structural genes)and/or which follow (3′, or downstream) the same. They include, inparticular, sequence regions which have a regulatory function. They canaffect transcription, RNA stability, RNA processing or else translation.Examples of regulatory sequences are, inter alia, promoters, enhancers,operators, terminators or translation enhancers.

The present invention furthermore relates to a gene structure containingat least one nucleotide sequence encoding a phospholipid:diacylglycerolacryltransferase and regulatory sequences which are linked operablytherewith and which govern the expression of the coding sequences in thehost cell.

Examples of suitable host cells are plant cells or algal cells, ormicroorganisms such as E. coli, yeast or filamentous fungi.

Operable linkage is understood as meaning the sequential arrangement of,for example, promoter, coding sequence, terminator and, if appropriate,further regulatory elements in such a way that each of the regulatoryelements can fulfill its intended function upon expression of the codingsequence. These regulatory nucleotide sequences can be of natural originor else be obtained by chemical synthesis. A suitable promoter is, inprinciple, any promoter capable of governing gene expression in the hostorganism in question.

The following may be mentioned by way of example: the cauliflower mosaicvirus promoter CaMV35S (Frank et al., 1980, Cell, 21: 285) or the B.napus napin promoter (Stalberg et al., 1993, Plant Molecular Biology,23:671-683). In accordance with the invention, this promoter may also bea chemically inducible promoter by means of which the expression of thegenes which it controls can be controlled, in the host cell, at aparticular point in time. Other advantageous promoters are those whichpermit tissue-specific expression, preferably seed-specific expression.Examples which may be mentioned are the following promoters: the USPpromoter (Bäumlein et al., 1991, Mol. Gen. Genet., 225 (3): 459-467, theoleosin promoter (WO 98/45461) or the B4 promoter from legumes (LeB4;Bäumlein et al., 1992, Plant Journal, 2 (2): 233-239.

A gene structure is generated by fusing a suitable promoter to at leastone nucleotide sequence according to the invention, using customaryrecombination and cloning techniques as are described, for example, inT. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY(1989) or Kaiser et al., 1994, Methods in Yeast Genetics, Cold SpringHarbor Laboratory Press or Guthrie et al., 1994, Guide to Yeast Geneticsand Molecular Biology, Methods in Enzymology, Academic Press.

Adaptors or linkers may be attached to the fragments to connect the DNAfragments with one another.

In addition, the present invention relates to a vector containing atleast one nucleotide sequence of the above-described type encoding aphospholipid:diacylglycerol acyltransferase, regulatory nucleotidesequences linked operably to the former, and additional nucleotidesequences for the selection of transformed host cells, for replicationwithin the host cell or for integration into the relevant host cellgenome. Moreover, the vector according to the invention may contain agene structure of the abovementioned type.

Vectors which are suitable are, for example, those which are replicatedin microorganisms or plants. The following enumeration is not limitingfor the present invention: PGEX (Pharmacia Biotech, Inc.; Smith et al.,1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), pRIT5(Pharmacia, Piscataway, N.J.) with glutathione S-transferase (GTS),maltose binding protein or proteinA, pTrc(Amann et al., 1988, Gene 69:301-315), pET vectors (Sudier et al., Genen Expression Technology,Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990:60-89 and Strategene, Amsterdam, Netherlands), pYepSec1 (Baldari et al.,1987, Embo J. 6: 229-234), pMFa (Kurjan et al., 1982, Cell 30: 933-943),pJRY88 (Schultz et al., 1987, Gene 54: 113-123), pYES derivatives(Invitrogen Corporation, San Diego, CA) or vectors for use infilamentous fungi are described in: van den Hondel, C. A. M. J. J. &Punt, Pl. (1991) “Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of Fungi, J. F.Peberdy et al., eds., p. 1-28, Cambridge University, Press: Cambridge.Examples of plant expression vectors are also found in Becker, D., etal. (1992) “New plant binary vectors with selectable markers locatedproximal to the left border, Plant Mol Biol 20: 1195-1197 or in Bevan, MW. (1984) “Binary Agrobacterium vectors for plant transformation”, Nucl.Acid. Res. 12: 8711-8721. As an alternative, insect cell expressionvectors may also be used, for example for expression in Sf 9 cells. Theyare, for example, the vectors of the pAc series (Smith et al. (1983) MolCell Biol 3:2156-2165) and vectors of the pVL series (Lucklow andSummers (1989) Virology 170:31-39). Further expression vectors are pCDM8and pMT2PC, which are mentioned in: Seed, B. (1987) Nature 329:840 orKaufman et al., (1987) EMBO J. 31 6: 187-195. Promoters preferably to beused are of viral origin, such as, for example, promoters of polyomavirus, adenovirus 2, cytomegalovirus or simian virus 40. Furtherprokaryotic and eukaryotic expression systems are mentioned in Chapters16 and 17 in Sambrook et al., Molecular Cloning: A Laboratory Manual2^(nd), ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

Using the nucleic acid sequences according to the invention, it is alsopossible to synthesize suitable probes or else primers which can be usedfor amplifying and isolating analogous genes from other organisms, forexample with the aid of PCR technology.

In addition, the present invention relates to the amino acids with PDATactivity which are derived from the nucleic acids. These also includeisoenzymes of PDAT. Isoenzymes are understood as meaning enzymes whichhave the same or a similar substrate specificity and/or catalyticactivity, but a different primary structure. Also encompassed inaccordance with the invention. are modified forms of PDAT. In accordancewith the invention, these are understood as meaning enzymes in whichmodifications are present in the sequence, for example at the N and/or Cterminus of the polypeptide or in the region of conserved amino acids,but without adversely affecting the function of the enzyme. Thesemodifications can be carried out by methods known per se in the form ofamino acid substitution.

A particular embodiment of the present invention also encompasses theuse of variants of the PDAT according to the invention whose activity isweakened or enhanced in comparison with the original protein inquestion, for example owing to amino acid substitution. The same appliesto the stability of the enzyme according to the invention in cells whichare more or less susceptible to, for example, degradation by proteases.

Also subject of the present invention are polypeptides with the functionof a PDAT whose amino acid sequence is modified in such a way that theyare desensitive to regulatory compounds, for example to cataboliteswhich regulate their activity (feedback desensitive).

The present invention furthermore also includes the transfer of anucleic acid sequence as shown in SEQ ID No. 1 or part thereof whichencodes a PDAT or an allele, homolog or derivative thereof into a hostsystem. The transfer of a gene construct or vector according to theinvention into a suitable host system is also included. The transfer offoreign genes into the genome of the plant is referred to astransformation. Generally customary methods for the transformation andregeneration of plants from plant tissues or plant cells are exploitedfor transient or stable transformation.

Suitable methods are protoplast transformation by polyethyleneglycol-induced DNA uptake, the biolistic method using the gene gun (whatis known as the particle bombardment method), electroporation,incubation of dry embryos in DNA-containing solution, microinjection andthe agrobacterium-mediate gene transfer. The abovementioned methods aredescribed, for example, in B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,edited by S. D. Kung and R. Wu, Academic Press (1993) 128-143, inPotrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42, (1991),205-225, Moloney et al., 1992, Plant Cell Reports, 8: 238-242, Mlynarovaet al., 1994, Plant Cell Report, 13: 282-285 and Bell et al., 1999, InVitro Cell. Dev. Biol.-Plant., 35 (6): 456-465.

The construct to be expressed is preferably cloned into a vector whichis suitable for transforming Agrobacterium tumefaciens, for examplepBinI9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteriatransformed with such a vector can then be used in a known manner fortransforming plants, in particular crop plants, such as, for example,tobacco plants, for example by bathing scarified leaves or leaf sectionsin an agrobacterial solution and subsequently growing them in suitablemedia. The transformation of plants with Agrobacterium tumefaciens isdescribed, for example, by Höfgen and Willmitzer in Nucl. Acid Res.(1988), 16, 9877 or is known from, inter alia, F. F. White, Vectors forGene Transfer in Higher Plants; in Transgenic Plants, Vol. 1,Engineering and Utilization, edited by S. D. Kung and R. Wu, AcademicPress, 1993, 5, 15-38.

Agrobacteria transformed with a vector according to the invention canlikewise be used in the manner known per se for transforming plants suchas laboratory plants such as Arabidopsis or crop plants such as cereals,maize, oats, rye, barley, wheat, soybean, rice, cotton, sugar beet,canola, sunflower, flax, hemp, potato, tobacco, tomato, carrot,capsicum, oilseed rape, tapioca, cassava, arrowroot, tagetes, alfalfa,lettuce and the various tree, nut and vine species, in particularoil-containing crop plants such as soybean, peanut, castor-oil plant,sunflower, maize, cotton, flax, oilseed rape, coconut, oil palm,safflower (Carthamus tinctorius) or cocoa bean, for example by bathingscarified leaves or leaf segments in an agrobacterial solution andsubsequently growing them in suitable media.

The genetically modified plant cells can be regenerated by all methodswhich are known to the skilled worker. Suitable methods can be found inthe abovementioned publications by S. D. Kung and R. Wu, Potrykus orHöfgen and Willmitzer.

The present invention also encompasses host organisms into which atleast one of the abovementioned nucleotide sequences encoding aphospholipid:diacylglycerol acyltransferase and/or a corresponding geneconstruct and/or a corresponding vector of the abovementioned type hasbeen transferred. In addition, the host organisms may additionally alsocontain nucleotide sequences which encode enzymes which participate inthe synthesis of unusual fatty acids. Again, these nucleotide sequencesmay be of natural origin or generated synthetically. Moreover, they canbe genetically modified and present in a comparable gene constructand/or vector of the abovementioned type. In this context, it isfeasible that the nucleotide sequences encoding aphospholipid:diacylglycerol acyltransferase and enzymes for thesynthesis of unusual fatty acids are present in one gene constructand/or vector or else in different gene constructs or vectors. In thesetransgenic host organisms according to the invention, in which theproduction of unusual fatty acids is enhanced in comparison with acorresponding, untransformed host organism, the nucleotide sequenceencoding a phospholipid:diacylglycerol acyltransferase is present in anincreased quantity, at least 2 copies and/or is expressed to a higherdegree starting from upstream regulatory sequences. Furthermore, thephospholipid:diacylglycerol acyltransferase activity in a host organismwhich has been transformed in accordance with the invention may beincreased as compared to the untransformed wildtype, which is firstlydue to an increased amount of enzymes which are present in the cell orelse, inter alia, due to a phospholipid:diacylglycerol acyltransferasewhose catalytic activity has been modified. The regulation of the enzymeactivity may furthermore also be modified.

In accordance with the invention, the host organisms, in principle, takethe form of all organisms which are capable of synthesizing fatty acidsand in the present context specifically unusual fatty acids, such aspolyunsaturated, longer-chain unsaturated or conjugated fatty acids, orof organisms which are suitable for the expression of recombinant genes.They are preferably plants or plant cells, preferably useful plants ortheir cells. Plants which are preferred in accordance with the inventionare Arabidopsis, Asteraceae such as Calendula, or crop plants such assoybean, peanut, castor-oil plant, sunflower, maize, cotton, flax,linseed, thistles, oilseed rape, coconut, oil palm, safflower (Carthamustinctorius) or cocoa bean. However, microorganisms such as fungi, forexample the genus Mortierella, Saprolegnia or Pythium, bacteria such asthe genus Escherichia, yeasts such as the genus Saccharomyces,cyanobacteria, ciliates, algae or protozoans such as dinoflagellates orCrypthecodinium, are also feasible.

Organisms which are preferred are those which are naturally capable ofsynthesizing oils in substantial amounts, for example fungi such asMortierella alpina, Pythium insidiosum or plants such as soybean,oilseed rape, coconut, oil palm, safflower, castor-oil plant, Calendula,peanut, cocoa bean, sunflower, or yeasts such as Saccharomycescerevisiae.

Utilizable host cells are furthermore mentioned in: Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990).

The host organisms are grown or cultured in a manner with which theskilled worker is familiar. As a rule, microorganisms are grown in aliquid medium which contains a carbon source, usually in the form ofsugars, a nitrogen source, usually in the form of organic nitrogensources such as yeast extract or salts such as ammonium sulfate, traceelements such as iron salts, manganese salts, or magnesium salts and, ifappropriate, vitamins at temperatures of between 0° C. and 100° C.,preferably between 10° C. and 60° C., while passing in oxygen gas. Inthis context, the pH value of the liquid medium can be kept constant,that is to say regulated during the culture period, or not. Culturingcan be carried out batchwise, semibatchwise or continuously. Nutrientscan be introduced at the beginning of the fermentation or else fedsemicontinuously or continuously.

To generate transgenic plants, binary vectors in Agrobacteriumtumefaciens or Escherichia coli are made use of, for example. For thetransformation, a 1:50 dilution of an overnight culture of a transformedagrobacteria colony in Murashige-Skoog medium (MS medium; Murashige andSkoog, 1962, Physiol. Plant, 15: 473) supplemented with 3% of sucrose(3MS medium) is made use of. Petioles or hypocotyls of freshlygerminated sterile plants (in each case approx. 1 cm²) are incubated for5-10 minutes in a 1:50 agrobacteria dilution in a Petri dish. This isfollowed by coincubation in the dark for 3 days at 25° C. on 3MS mediumsupplemented with Bacto agar. After 3 days, culturing was continued at16 hours light/8 darkness, and continued in a weekly rhythm on MS mediumsupplemented with Claforan (cefotaxime-sodium), antibiotic,benzylaminopurine (BAP) and glucose. The young shoots are transferred toMS medium supplemented with 2% sucrose (2MS medium), Claforan and Batchagar. If no roots develop, the growth hormone 2-indolebutyric acid isadded to the medium to induce rooting. Regenerated shoots are obtainedon 2MS medium supplemented with antibiotic and Claforan; after theshoots have rooted, they are transferred into soil and, after havingbeen grown for 2 weeks, grown in a controlled-environment cabinet or inthe greenhouse, allowed to flower, and mature seeds are harvested andtheir fatty acid content is analyzed.

In accordance with the invention, the storage lipids (triacylglycerols,TAGs) of the transgenic organisms according to the invention show anincreased content of unusual fatty acids, such as polyunsaturated fattyacids, long-chain polyunsaturated fatty acids or conjugated fatty acids.In this context, the content of these fatty acids is increased incomparison with the fatty acid content which is normally found in thestorage lipids of these plants.

After the transgenic organisms according to the invention have beengrown, the lipids are obtained in the customary manner. To this end, theorganisms can first be disrupted after they have been harvested or elseused directly. The lipids are advantageously extracted using suitablesolvents such as apolar solvents such as hexane or ethanol, isopropanolor mixtures such as hexane/isopropanol, phenol/chloroform/isoamylalcohol, at temperatures of between 0° C. and 80° C., preferably between20° C. and 50° C. As a rule, the biomass is extracted with an excess ofsolvent, for example a 1:4 excess of solvent to biomass. The solvent issubsequently removed, for example via distillation. Alternatively, theextraction may be carried out with supercritical CO₂. After theextraction, the remainder of the biomass can be removed for example byfiltration.

The crude oil thus obtained can subsequently be purified further, forexample by removing cloudiness by treatment with polar solvents such asacetone or chloroform, followed by filtration or centrifugation. Furtherpurification through columns is also possible. To obtain the free fattyacids from triglycerides, the latter are hydrolzyed in the customarymanner.

The invention thus also relates to unusual polyunsaturated, longer-chainpolyunsaturated or conjugated fatty acids and to triglycerides with anincreased content of these fatty acids which have been produced by theabovementioned procedures, and to their use for the production of foods,animal feeds, cosmetics or pharmaceuticals. To this end, the former areadded to the foods, the animal feeds, the cosmetics or thepharmaceuticals in customary amounts.

In one variant of the present invention Arabidopsis thaliana plants withthe nucleotide sequence as shown in SEQ ID No. 1 (AB006704) weretransformed. These plants are subsequently analyzed for their novelcharacteristics:

Northern blot analyses

T2 plants which had been transformed with a control vector or with avector containing the gene AB006704 were employed for the RNAextraction. Since the T2 seedlings revealed segregation with regard tothe inserted gene, kanamycin was employed to eliminate untransformedseedlings. T2 seedlings of A. thaliana C24 transformed with the controlvector, and T2 seedlings of 3 different 35S-AB006704 A. thaliana cv.Columbia transformants which had survived after germinating on kanamycinplates, were grown in liquid culture and used for the RNA extraction.RNA was prepared from leaves and roots and separated in a Northern blot(FIG. 1 a). The expression of the Arabidopsis AB006704 gene the leavesand roots of A. thaliana C24 (transformed with the control vector) wasbarely detectable. The expression of the AB006704 gene was clearlyvisible in all of the 3 35S-AB006704 transformants, not only in theleaves but also in the roots, the highest expression level beingobserved in roots. AB006704 was expressed at the highest expression ratein transformant no. 1-1-6, the transformants 1-3b-44 and 1-2-13 showingbands with approximately 70% and 25%, respectively, of the intensity ofthe hybridization band of transformant 1-1-6.

PDAT enzyme assay

PDAT activity was determined in microsomal preparations of leaves androots of T2 plants of A. thaliana C24 (transformed with the controlvector) and of T2 plants of 3 different 35S-AB006704 transformants of A.thaliana cv. Columbia. Plants used for the preparation of microsomeswere grown under the same conditions as plants which were used for thedetection of AB006704 MRNA by Northern blot analyses. In earlierexperiments (Dahlqvist et al., 2000, Proc. Natl. Acas. Sci., USA,97:6487-6492; data not shown), PC with ricinoleic acid in position sn-2was, in most cases, one of the best substrates for PDAT-catalyzedreactions.

The content of de novo-synthesized 1-OH-TAG generally represents theexpression pattern of the AB006704 gene in plant material which was usedfor microsomal preparation (FIGS. 1 b and 1 c). Microsomes oftransformant 1-1-6 (with the highest level of AB006704 gene expression)produced more TAG then microsomes of transformant 1-3b-44 (mediumexpression level). The microsomes of transformant 1-3b-44 synthesizedmore TAG than microsomes of transformant 1-2-13 (low expression level).Moreover, the microsomes of transformant 1-2-13 produced more TAG thanmicrosomes which had been transformed with the blank control vector forcontrol purposes.

AB006704 encodes an enzyme with PDAT activity

To demonstrate that the formation of ¹⁴C-labeled TAG from the previousexperiment takes place via a PDAT-catalyzed reaction with PC as fattyacid donor and DAG as acyl acceptor, sn-1-oleoyl-sn-2-epoxy-DAG wasemployed as acyl acceptor and sn-1-oleoyl-sn-[¹⁴C]ricinoleoyl-PC as acyldonor in this experiment. Enzyme assays were carried out with the samemicrosomal preparation of leaves of T2 plants of A. thaliana C24(transformed with the control vector) and T2 plants of 3 differenttransformants of A. thaliana cv. Columbia (with the AB006704 gene underthe control of the 35S promoter) as was the case in the previousexperiment. The de novo synthesis of TAG molecules containing both ofthe [¹⁴C]-ricinoleoyl and -vernoloyl groups (FIG. 2) clearly demonstratethe expression pattern of the AB006704 gene in plant material which hadbeen used for the preparation of microsomes (FIG. 1 a). The dataobtained clearly demonstrates that the transgenic PDAT gene is capableof exploiting fatty acids from the sn-2 position of PC toacyl-sn-1-oleoyl-sn-2-epoxy-DAG for the formation of TAG. Here, thehydroxy- and epoxy-fatty acids in the transgenic plants containing thegene AB006704 are incorporated better into triacylglycerides (TAGs) thanin the control plants. Accordingly, it has been demonstrated that thegene AB006704 encodes an enzyme with PDAT activity and is capable ofexploiting PC as intermediate acyl donor and DAG as acyl acceptor in anacyl-CoA-independent reaction for the formation of TAG.

Substrate specificity

To study the substrate specificity (FIG. 3) of the A. thaliana proteinencoded by the AB006704 gene, a microsomal preparation from leaves oftransformant 1-1-6, which had been grown in liquid culture, was used forthe assay. Protein AB006704 shows a higher activity toward PC withunsaturated fatty acids in position sn-2 than toward PC with saturatedfatty acids. Among the 18-C fatty acids, PC with linolenic acid (18:3)in position sn-2 was the best substrate, while stearic acid (18:0) wastransferred least. Moreover, for example, erucic acid (22:1) wastransferred much less than oleic acid (18:1); arachidonic acid (20:4)was transferred at approximately the same rate as linolenic acid (18:3),even though arachidonic acid has one more double bond than linolenicacid.

Ricinoleic acid, which contains a hydroxyl group at position 12, wastransferred to DAG with the highest efficacy of all acyl groups tested.Also, vernolic acid which contained an epoxy group, was transferred fromthe sn-2 position of PC to DAG with approximately twice the efficiencyof the corresponding linoleic acid. Moreover, the A. thaliana PDAT whichwas studied showed differences in specificity toward phospholipids withhead groups of different polarity. Phosphatidylethanolamine (PE) wasutilized somewhat better than phosphatidylcholine (PC). The transfer of10:0, or 18:1, by PE was approximately 1.5 times more rapid than in thecase of PC. In contrast, phosphatidylic acid (PA) was a worse substratethan PC. The transfer of oleic and ricinoleic acid from position sn-2 ofPA was 3 to 5 times less efficient than in the case of PC. Acylcompounds of the acceptor have the same effect on the activity of the A.thaliana PDAT which has been studied. For example,sn-1-oleoyl-sn-2-epoxy-DAG is a somewhat better acceptor thandioleoyl-DAG (FIG. 1B and FIG. 2).

This means that not only the chain length but also the number of doublebonds and likewise the functional group in the vicinity of the headgroup has an effect on enzyme activity.

The present invention is illustrated hereinbelow by further exampleswhich, however, do not limit the invention:

1. General methods

The isolation of plasmid DNA from bacteria or plants and all techniquesregarding restriction, treatment with Klenow and alkaline phosphatase,electrophoresis, transformation, sequencing, RNA analyses or PCR werecarried out as described by Sambrook et al. (Molecular cloning. Alaboratory manual (1989) Cold Spring Harbour Laboratory Press).

2. Preparation of gene constructs and vectors

The nucleotide sequence AB006704 (SEQ ID No. 1), which encodes a PDATenzyme from Arabidopsis thaliana (SEQ ID No. 2), was placed under thecontrol of the 35S promoter.

Using restriction digestion, a NotI fragment was isolated from theligation product and cloned into the binary vector pART27 (Lee et al.,1998, Science, 280:915-918) downstream of the napin promoter (forseed-specific expression). The vector pART27 without the nucleotidesequence encoding A. thaliana PDAT acted as a control vector.

3. Transformation of Arabidopsis thaliana

The above-described vector pART27 containing AB006704 was transferredinto A. thaliana cv. Columbia plants by means of vacuum infiltration(Bent et al., 2000, Plant Physiol. 124, (4): 1540-1547). A correspondingcontrol vector was transformed into an A. thaliana cv. C24. T1 seedswere seeded on ⅓ MS plates containing 1% sucrose and 50 μg/ml kanamycin.Grown seedlings were transplanted into soil, and T2 seeds wereharvested.

To verify whether the gene AB006704 encodes the PDAT enzyme, constructsfor direct expression of the AB006704 gene in plants were generated. Tothis end, the gene was cloned into a binary vector pART27, eitherdownstream of the CaMV 35S promoter or the B. napus napin promoter, andArabidopsis thaliana (cv. Columbia) was transformed by means of vacuuminfiltration. T2 seedlings were analyzed for PDAT activity, and theexpression of the gene AB006704 was studied with the aid of Northernblot analyses.

4. Microsomal preparation

T2 seeds of A. thaliana cv. C24 transformed with the blank vector and T2seeds of A. thaliana cv. Columbia transformed with a vector containingAB006704 under the control of the 35S promoter were seeded on ⅓ MSplates containing 1% sucrose and 50 μg/ml kanamycin. Untransformed A.thaliana seeds (cv. Columbia) were seeded on plates without kanamycin.After 10 days, the seedlings which had grown were transferred intoculture vessels containing liquid containing ½ MS containing 1% sucroseand incubated for 27 days at 23° C. in the light on a shaker. Microsomesof leaves and roots of the A. thaliana seedlings which had grown inliquid culture were prepared using the method of Stobart and Stymne(Biochemical Journal, 1985, 232 (1): 217-221).

5. Preparation of lipid substrates

Radiolabeled ricinoleic acid (12-hydroxy-9-octadecenoic acid) andvernolic acid (12,13-epoxy-9-octadecenoic acid) were synthesizedenzymatically from [1-¹⁴C]oleic acid and [1-¹⁴C]-linoleic acid,respectively, by incubation with microsomal preparations from Ricinuscommunis or Crepis palaestina seeds, respectively (Bafor et al., 1991,Biochem. J. 280:507-514). Radiolabeled crepenynic acid(9-octadecen-12-ynoic acid) were synthesized enzymatically from[-1-¹⁴C]-linolic acid by incubation with microsomal preparations fromCrepis alpina (Lee et al., 1998, Science, 280: 915-918). Radiolabeled10:0, 18:0, 18:1, 18:2, 18:3, 20:1 and 20:4 fatty acids are commerciallyavailable. The synthesis of phosphatidylcholines (PC),phosphatidylethanolamines (PE) and phosphatidylic acid (PA) with¹⁴C-labeled acyl groups in position sn-2 was carried out either byenzymatic acylation (Banas et al., 1992, Plant Science, 84: 137-144) orsynthetic acylation. Sn-1-oleoyl-sn2-epoxy-DAG was prepared from Crepispalaestina triacylglycerols by treatment with lipase and separation viathin-layer chromatography.

6. Enzyme assay

Aliquots of crude microsomal fractions (corresponding to 12 nmolmicrosomal PC) from developing plant seeds, were lyophilized overnight.¹⁴C-labeled substrate lipids (2.5 nmol of PC, PE or PA with ¹⁴C-labeledacyl groups in position sn-2 and 1.5 nmol of dioleoyl- orsn1-oleoyl-sn2-epoxy-DAG) which had been dissolved in benzene were thenadded to dried microsomes. The benzene was evaporated by passing in N₂,whereby the lipids were brought into direct contact with the membranes,and 0.1 ml of 50 mM potassium phosphate (pH 7.2) was added. Thesuspension was mixed thoroughly and incubated at 30° C. over the periodstated of up to 60 min. The lipids were extracted from the reactionmixture using chloroform and separated. by thin-layer chromatographyusing silica gel 60 plates (Merck) in hexane/diethyl ether/ acetic acid(35:70:1.5 v/v) using silica gel 60 plates (Merck). The radioactivelipids were visualized on the plates via autoradiography (InstantImager, Packard, USA) and quantified.

7. Growth experiments

Untransformed A. thaliana seedlings (cv. Columbia) and T2 seedlings ofA. thaliana (cv. Columbia) transformed with the vector containingAB006704 under the control of the 35S promoter (plant 1-1-6 with thegene AB006704 with the highest expression) were seeded on 4 different ⅓MS plates containing 1% sucrose (one half of the plates were seeded withcontrol seedlings, while the other half was seeded with transformedseedlings) and grown for 17 days at 23° C. with exposure to light. Thefresh weight of each seedling (approximately 50 seedlings of each type)was determined. Controls and transformed seedlings for each plate werecollected and employed for lipid analysis.

8. Fatty acid content and lipid analysis

The fatty acid in A. thaliana wild-type (cv. Columbia) seedlings and incorresponding transformants was determined by extracting the lipidsfollowing the method of Bligh & Dyer (1959, Can. J. Biochem. Physiol.,37, 911-917) followed by methylation with 2% H₂SO₄ in dried methanol (60minutes at 90° C.). Lipids in the Arabidopsis seedlings (2-3 mg/sample)were methylated directly with 2 ml of 2% strength (v/v) H₂SO₄ in driedmethanol (90 minutes at 90° C.). The methyl esters were extracted withhexane and analyzed via GLC using “Chrompack” capillary columns (WCOTfused-silica column 50 m×0.32 mm ID coated with CD wax 58-CB DF=0.2). Toquantify the fatty acid content, methylheptadecanoic acid was used asthe internal standard.

Key to figures:

The present invention will additionally be illustrated hereinbelow withreference to the figures:

FIG. 1A shows an RNA (Northern blot) analysis of total RNA from leavesand roots of A. thaliana C24 control plants transformed with the blankcontrol vector and three different A. thaliana plants transformed withthe vector containing a nucleotide sequence as shown in SEQ ID No. 1(PDAT) under the control of the 35S promoter.

FIG. 1B and FIG. 1C show the conversion ofsn-1-oleoyl-sn-2-[¹⁴C]-ricinoleyl-PC during incubation for 1 hour withmicrosomes (and unlabeled sn-1-oleoyl-sn-2-oleoyl-DAG) from leaves (FIG.1B) and roots (FIG. 1C) of A. thaliana C24 control plants and threedifferent A. thaliana plants transformed with the vector containing anucleotide sequence as shown in SEQ ID No. 1 (PDAT) under the control ofthe 35S promoter. The average radioactivity present in the denovo-synthesized 1-OH-TAG is shown in the autoradiograph as apercentage.

FIG. 2 shows the in vitro synthesis of TAG containing a vernoloyl and a[¹⁴C]-recinoleoyl group in microsomes of leaves of A. thaliana C24control plant and three different A. thaliana plants transformed withthe vector containing a nucleotide sequence as shown in SEQ ID No. 1(PDAT) under the control of the 35S promoter. The substrates added aresn-1-oleoyl-sn-2-[¹⁴ ]-epoxy-DAG andsn-1-oleoyl-sn-2-[¹⁴C]-ricinoleoyl-PC. The average radioactivity presentin the de novo-synthesized 1-OH-TAG and 1-OH-1-epoxy-TAG is shown in theautoradiographs as a percentage.

FIG. 3 shows the substrate specificity of the protein with PDAT activitywhich is encoded by the nucleotide sequence as shown in SEQ ID No. 1.Microsomal preparations of leaves of the A. thaliana transformant 1-1-6transformed with the vector containing a nucleotide sequence as shown inSEQ ID No. 1 (PDAT) under the control of the 35S promoter were employed.Dioleoyl-DAG together with sn-1-oleoyl-sn-2-[¹⁴C]-fatty acidphospholipids (PC, PE, PA) were employed as substrate (18:0-PC, 20:4-PC,22:1-PC, 10:0-PC and 10:0-PE with 16:0 in position sn-1). Relativeenzyme activities of PADAT towards various substrates are shown, theactivity towards 18:1-PC being designated 1 (20:4 is arachidonic acid).

Also appended is a sequence listing containing SEQ ID No. 1 and SEQ IDNo. 2 for a PDAT enzyme from Arabidopsis thaliana.

1. A method for production of plant storage lipids containingpolyunsaturated fatty acids comprising providing an enzyme mixturecontaining at least one enzyme with phospholipid:diacylglycerolacyltransferase activity.
 2. The method of claim 1, wherein the enzymemixture further contains at least one further activity of a hydroxylase,epoxygenase, acetylenase, desaturase, elongase, conjugase,trans-desaturase, isomerase or combination thereof.
 3. The method ofclaim 1, wherein the enzyme mixture further contains desaturase activityand elongase activity.
 4. The method of claim 1, wherein thepolyunsaturated fatty acids comprise long-chain polyunsaturated fattyacids.
 5. The method of claim 1, wherein the polyunsaturated fatty acidscomprise one or more of gamma-linolenic acid, arachidonic acid,gamma-limolenic acid, eicosapentaenoic acid, stearidonic acid ordocosahexaenoic acid.
 6. The method of claim 1, wherein the at least oneenzyme is encoded by a nucleotide sequence which is capable ofreplication, is present in a plant cell in at least 2 copies or containsregulatory sequences that bring about an at least 2-fold increase ingene expression or enzyme activity.
 7. The method of claim 6, whereinthe nucleotide sequence is encoded chromosomally or extrachromosomally.8. The method of claim 6, wherein the nucleotide sequence is derivedfrom plants.
 9. The method of claim 6, wherein the nucleotide sequenceis derived from Arabidopsis thaliana.
 10. The method of claim 1, whereinthe at least one enzyme comprises the amino acid sequence of SEQ ID No.2.
 11. The method of claim 1, wherein the at least one enzyme or a partthereof is encoded by the nucleotide sequence of SEQ ID No. 1 or allelesthereof.
 12. The method of claim 1, wherein the polyunsaturated fattyacids contain fatty acids with conjugated double bonds.
 13. The methodof claim 1, wherein the polyunsaturated fatty acids comprise fatty acidswith a chain length of at least 14 carbon atoms and having at least 3double bonds.
 14. The method of claim 1, wherein the polyunsaturatedfatty acids comprise fatty acids not naturally found in plants.
 15. Amethod for producing polyunsaturated acids from a plant comprising:increasing a phospholipid:diacylglycerol acyltransferase activity ofsaid plant; and isolating the polyunsaturated fatty acids.
 16. Themethod of claim 15, wherein increasing comprises increasing the copynumber of a gene that encodes a phospholipid:diacylglycerolacyltransferase enzyme.
 17. The method of claim 15, wherein increasingcomprises increasing the catalytic or regulatory activity of one or moreenzymes involved in synthesis of fatty acids.
 18. The method of claim15, wherein increasing comprising transforming said plant with anucleotide sequence.
 19. The method of claim 18, wherein the nucleotidesequence comprises SEQ ID No. 1 or a homolog or allele thereof.
 20. Themethod of claim 19, wherein the homolog has a sequence which is at least60% identical to said nucleotide sequence.